We previously reported that human primary macrophage have approximately 100 times lower dNTP concentrations (20-40nM) than activated CD4+ T cells (2~5M). The limited dNTP pools impose a strong restriction on HIV reverse transcription in macrophages. Several recent papers in Nature reported that Vpx, a co-packaged accessory protein of HIV-2 and SIVs, enhances viral infectivity in macrophages by degrading a myeloid cell specific, host viral restriction factor, SAMHD1, which suppresses HIV-1 replication in macrophages. Two more follow-up studies revealed that SAMHD1 is a dGTP-dependent dNTP hydrolase, which can hydrolyze and inactivate cellular dNTPs, suggesting that SAMHD1 restricts HIV-1 infection in nondividing macrophages by lowering cellular dNTP pools, which is logically consistent with our observation that the dNTP concentration in macrophages is extremely low. Indeed, our recently accepted report in Nature Immunology revealed that while SAMHD1 expression reduces cellular dNTP pools, Vpx efficiently elevates cellular dNTP concentrations by degrading SAMHD1 in macrophages, supporting that Vpx enhances HIV infectivity by promoting proviral DNA synthesis kinetics in myeloid cell types. We predict that cellular dNTP availability mechanistically influences three important events of HIV biology that occur during the early step of HIV life cycle: (1) Viral sensitivity to NRTIs: The EC50 for NRTI- mediated inhibition of virus replication in acutely infected macrophages is approximately 10-fold lower than that in T cells. We hypothesize that Vpx reduces viral sensitivity to NRTIs by increasing cellular dNTP pools while SAMHD1 expression enhances viral sensitivity to NRTIs by hydrolyzing cellular dNTPs, in macrophages, but not in activated CD4+ T cells. (2) Viral recombination frequency: HIV-1 RT pausing, which is induced by lowering dNTP concentration during DNA synthesis, initiates RNA template switching and recombination. Thus, we hypothesize that dNTP pool modulation induced by Vpx and SAMHD1 directly affects HIV-1 recombination frequency in macrophages, but not in T cells. (3) Incorporation of mutagenic noncanonical dUTP during proviral DNA synthesis: We have shown that there is a 60-fold disparity between levels of dTTP (low) and the noncanonical dUTP in macrophages, but not in T cells. We further showed that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation (which can be mutagenic). Thus we hypothesize that Vpx, which elevates cellular dNTP levels (including dTTP, but not dUTP), can minimize the incorporation of potentially mutagenic dUTP in macrophages. We will investigate the structural and enzymological features of the SAMHD1 active site involved in dNTP substrate specificity.